STELLATE GANGLION BLOCK REVERSES PHSML-INDUCED VASCULAR HYPOREACTIVITY THROUGH INHIBITING AUTOPHAGY-MEDIATED PHENOTYPIC TRANSFORMATION OF VSMCs.

ABSTRACT: Posthemorrhagic shock mesenteric lymph (PHSML) return-contributed excessive autophagy of vascular smooth muscle cells (VSMCs) is involved in vascular hyporeactivity, which is inhibited by stellate ganglion block (SGB) treatment. The contractile phenotype of VSMCs transforms into a synthetic phenotype after stimulation with excessive autophagy. Therefore, we hypothesized that SGB ameliorates PHSML-induced vascular hyporeactivity by inhibiting autophagy-mediated phenotypic transformation of VSMCs. To substantiate this hypothesis, a hemorrhagic shock model in conscious rats was used to observe the effects of SGB intervention or intravenous infusion of the autophagy inhibitor 3-methyladenine (3-MA) on intestinal blood flow and the expression of autophagy- and phenotype-defining proteins in mesenteric secondary artery tissues. We also investigated the effects of intraperitoneal administration of PHSML intravenous infusion and the autophagy agonist rapamycin (RAPA) on the beneficial effect of SGB. The results showed that hemorrhagic shock decreased intestinal blood flow and enhanced the expression of LC3 II/I, Beclin 1, and matrix metalloproteinase 2, which were reversed by SGB or 3-MA treatment. In contrast, RAPA and PHSML administration abolished the beneficial effects of SGB. Furthermore, the effects of PHSML or PHSML obtained from rats treated with SGB (PHSML-SGB) on cellular contractility, autophagy, and VSMC phenotype were explored. Meanwhile, the effects of 3-MA on PHSML and RAPA on PHSML-SGB were observed. The results showed that PHSML, but not PHSML-SGB, incubation decreased VSMC contractility and induced autophagy activation and phenotype transformation. Importantly, 3-MA administration reversed the adverse effects of PHSML, and RAPA treatment attenuated the effects of PHSML-SGB incubation on VSMCs. Taken together, the protective effect of SGB on vascular reactivity is achieved by inhibiting excessive autophagy-mediated phenotypic transformation of VSMCs to maintain their contractile phenotype.

Analysis and Identification of Ferroptosis-Related Gene Signature for Acute Lung Injury.

BACKGROUND: Recent studies have shown that ferroptosis is involved in the evolution of acute lung injury (ALI), a serious respiratory pathological process leading to death. However, the regulatory mechanisms underlying ferroptosis in ALI remain largely unknown. The current study analyzed and identified a ferroptosis-related gene signature for ALI. METHODS: Key genes associated with ferroptosis in ALI were identified by bioinformatics analysis. GSE104214, GSE18341, and GSE17355 datasets were downloaded from the Gene Expression Omnibus database. The signature genes were screened by least absolute shrinkage and selection operator (LASSO) regression, and the key genes of ALI were screened by weighted correlation network analysis (WGCNA), followed by immune infiltration analysis and functional enrichment analysis. In addition, mRNA expression of key genes in the lungs of mice with hemorrhagic shock and sepsis was verified. RESULTS: A total of 2132 differential genes were identified by various analyses, and nine characteristic genes were detected using Lasso regression. We intersected nine signature genes with WGCNA module genes and finally determined four key genes (PROK2, IL6, TNF, SLC7A11). All four key genes were closely correlated with immune cells and regulatory genes of ALI, and the expression of the four genes was significantly different in the lung tissues of hemorrhagic shock and sepsis models. Besides, the ferroptosis related molecules GPX4 and ACSL4 showed remarkable difference in these models. CONCLUSION: These results indicate that PROK2, IL6, TNF, SLC7A11 may be key regulatory targets of ferroptosis during ALI. This study proved that ferroptosis is a common pathophysiological process in three ALI models.

Excision of mesenteric lymph nodes alters gut microbiota and impairs social dominance in adult mice.

INTRODUCTION: Mesenteric lymph nodes (MLNs) are central in immune anatomy. MLNs are associated with the composition of gut microbiota, affecting the central system and immune system. Gut microbiota was found to differ among individuals of different social hierarchies. Nowadays, excision of MLNs is more frequently involved in gastrointestinal surgery; however, the potential side effects of excision of MLNs on social dominance are still unknown. METHODS: MLNs were removed from male mice (7-8 weeks old). Four weeks after MLN removal, social dominance test was performed to investigate social dominance; hippocampal and serum interleukin (IL)-1ß, IL-10, and tumor necrosis factor-alpha (TNF-α) were investigated; and histopathology was used to evaluate local inflammation of the ileum. The composition of the gut microbiota was then examined to understand the possible mechanism, and finally intraperitoneal injection of IL-10 was used to validate the effect of IL-10 on social dominance. RESULTS: There was a decrease in social dominance in the operation group compared to the control group, as well as a decrease in serum and hippocampal IL-10 levels, but no difference in serum and hippocampal IL-1ß and TNF-α levels, and no local inflammation of the ileum after MLN removal. 16S rRNA sequencing analysis showed that the relative abundance of the class Clostridia was decreased in the operation group. This decrease was positively associated with serum IL-10 levels. Furthermore, intraperitoneal injection of IL-10 in a subset of mice increased social dominance. CONCLUSIONS: Our findings suggested that MLNs contributed to maintaining social dominance, which might be associated with reduced IL-10 and the imbalance of specific flora in gut microbiota.

ESTROGEN ALLEVIATES POSTHEMORRHAGIC SHOCK MESENTERIC LYMPH-MEDIATED LUNG INJURY THROUGH AUTOPHAGY INHIBITION.

ABSTRACT: Background: Hemorrhagic shock-induced acute lung injury (ALI) is commonly associated with the posthemorrhagic shock mesenteric lymph (PHSML) return. Whether excessive autophagy is involved in PHSML-mediated ALI remains unclear. The relationship between estrogen treatment and PHSML or autophagy needs to verify. The current study will clarify the role of estrogen in reducing PHSML-mediated ALI through inhibition of autophagy. Methods: First, a hemorrhagic shock model in conscious rats was used to observe the effects of 17ß-estradiol (E2) on intestinal blood flow, pulmonary function, intestinal and pulmonary morphology, and expression of autophagy marker proteins. Meanwhile, the effect of PHSML and autophagy agonist during E2 treatment was also investigated. Secondly, rat primary pulmonary microvascular endothelial cells were used to observe the effect of PHSML, PHSML plus E2, and E2-PHSML (PHSML obtained from rats treated by E2) on the cell viability. Results: Hemorrhagic shock induced intestinal and pulmonary tissue damage and increased wet/dry ratio, reduced intestinal blood flow, along with pulmonary dysfunction characterized by increased functional residual capacity and lung resistance and decreased inspiratory capacity and peak expiratory flow. Hemorrhagic shock also enhanced the autophagy levels in intestinal and pulmonary tissue, which was characterized by increased expressions of LC3 II/I and Beclin-1 and decreased expression of p62. E2 treatment significantly attenuated these adverse changes after hemorrhagic shock, which was reversed by PHSML or rapamycin administration. Importantly, PHSML incubation decreased the viability of pulmonary microvascular endothelial cells, while E2 coincubation or E2-treated lymph counteracted the adverse roles of PHSML. Conclusions: The role of estrogen reducing PHSML-mediated ALI is associated with the inhibition of autophagy.

POSTHEMORRHAGIC SHOCK MESENTERIC LYMPH IMPAIRS SPLENIC DENDRITIC CELL FUNCTION IN MICE.

ABSTRACT: Dendritic cell (DC)-mediated immune dysfunction is involved in the process of severe hemorrhagic shock that leads to sepsis. Although post-hemorrhagic shock mesenteric lymph (PHSML) induces immune organs injuries and apoptosis, whether PHSML exerts adverse effects on splenic DCs remains unknown. In this study, we established a hemorrhagic shock model (40 ± 2 mm Hg for 60 min) followed by fluid resuscitation with the shed blood and equal Ringer's solution and drained the PHSML after resuscitation. At 3 h after resuscitation, we harvested the splenic tissue to isolate DCs using anti-CD11c immunomagnetic beads and then detected the necrotic and apoptotic rates in splenocytes and splenic DCs. We also detected the levels of TNF-α, IL-10, and IL-12 in the culture supernatants and surface marker expressions of MHC-II, CD80, and CD86 of splenic DCs following LPS stimulation for 24 h. Second, we purified the DCs from splenocytes of normal mice to investigate the effects of PHSML treatment on cytokine production and surface marker expression following LPS stimulation. The results showed that PHSML drainage attenuated LPS-induced cell death of splenocytes and DCs. Meanwhile, PHSML drainage enhanced the DC percentage in splenocytes and increased the TNF-α and IL-12 production by DCs and the expressions of CD80, CD86, and MHCII of DCs treated by LPS. Furthermore, PHSML treatment reduced the productions of TNF-α, IL-10, and IL-12 and the expressions of CD80 and CD86 in normal DCs after treatment with LPS. In summary, the current investigation demonstrated that PHSML inhibited the cytokine production and surface marker expressions of DCs stimulated by LPS, suggesting that PHSML plays an important role in hemorrhagic shock-induced immunosuppression through the impairment of DC function and maturation.

TLR2/TLR4-Enhanced TIPE2 Expression Is Involved in Post-Hemorrhagic Shock Mesenteric Lymph-Induced Activation of CD4+T Cells.

Purpose: Post hemorrhagic shock mesenteric lymph (PHSML) return contributes to CD4+ T cell dysfunction, which leads to immune dysfunction and uncontrolled inflammatory response. Tumor necrosis factor α induced protein 8 like-2 (TIPE2) is one of the essential proteins to maintain the immune homeostasis. This study investigated the role of TIPE2 in regulation of CD4+ T lymphocyte function in interaction of PHSML and TLR2/TLR4. Methods: The splenic CD4+ T cells were isolated from various mice (WT, TLR2-/-, TLR4-/-) by immunomagnetic beads, and stimulated with PHSML, normal lymphatic fluid (NML), respectively. Application of TIPE2-carrying interfering fragments of lentivirus were transfected to WT, TLR4-/-, and TLR2-/- CD4+ T cells, respectively. After interference of TIPE2, they were stimulated with PHSML and NML for the examinations of TIPE2, TLR2, and TLR4 mRNA expressions, proliferation, activation molecules on surface, and cytokine secretion function. Results: PHSML stimulation significantly upregulated TIPE2, TLR2, and TLR4 mRNA expressions, decreased proliferation, CD25 expression, and IFN-γ secretion, and increased the secretion ability of IL-4 in WT CD4+ T cells. TIPE2 silencing enhanced proliferative capacity, upregulated CD25 expression, and increased IFNγ secretion in CD4+ T cells. PHSML stimulated TLR2-/-CD4+ T or TLR4-/-CD4+ T cells of which TIPE2 were silenced. TLR2 or TLR4 knockout attenuated PHSML-induced CD4+ T cells dysfunction; PHSML stimulation of silent TIPE2-expressing TLR2-/-CD4+ T or TLR4-/-CD4+ T revealed that the coexistence of low TIPE2 expression with lack of TLR2 or TLR4 eliminated this beneficial effect. Conclusion: TIPE2 improves the PHSML-mediated CD4+T cells dysfunction by regulating TLR2/TLR4 pathway, providing a new intervention target following hemorrhagic shock-induced immune dysfunction.

Autophagy Is Involved in Stellate Ganglion Block Reversing Posthemorrhagic Shock Mesenteric Lymph-Mediated Vascular Hyporeactivity.

Objective: The aim of this study was to clarify the role of autophagy in stellate ganglion block (SGB) reversing posthemorrhagic shock mesenteric lymph (PHSML)-mediated vascular hyporeactivity. Methods: Hemorrhagic shock model in conscious rats was employed to observe the effects of SGB (0.2 ml of 0.25% ropivacaine hydrochloride hydrate) and autophagy inhibitor 3-methyladenine (3-MA; 30 mg/kg) on the vascular reactivity of second-order rat mesenteric arteries in vitro, while the effects of PHSML (1 ml/kg) and autophagy agonist rapamycin (Rapa, 10 mg/kg) on the beneficial effect of SGB were investigated. The cellular viability, contractility, and autophagy-related protein expressions in vascular smooth muscle cells (VSMCs) were detected following treatments of PHSML, PHSML obtained from the rats that underwent hemorrhagic shock plus SGB (PHSML-SGB), and PHSML plus 3-MA (5 mM), respectively. Results: Hemorrhagic shock significantly decreased the vascular reactivity to gradient norepinephrine (NE), which is reversed by the SGB treatment and 3-MA administration. On the contrary, PHSML intravenous infusion and Rapa administration inhibited the vascular contractile responses in rats that underwent hemorrhagic shock plus SGB treatment. PHSML treatment significantly inhibited the cellular viability and contractility in VSMCs, increased the expressions of LC3-II and Beclin 1, and decreased the expression of p62, along with opposite appearances in these indices following PHSML-SGB treatment. In addition, 3-MA counteracted the adverse roles of PHSML in these indices in VSMCs. Conclusion: SGB inhibits PHSML-mediated vascular hyporeactivity by reducing the excessive autophagy in VSMCs.

Stellate Ganglion Block Improves the Proliferation and Function of Splenic CD4 + T Cells Through Inhibition of Posthemorrhagic Shock Mesenteric Lymph-Mediated Autophagy.

Severe hemorrhagic shock leads to excessive inflammation and immune dysfunction, which results in high mortality related to mesenteric lymph return. A recent study showed that stellate ganglion block (SGB) increased the survival rate in rats suffering hemorrhagic shock. However, whether SGB ameliorates immune dysfunction induced by hemorrhagic shock remains unknown. The aim of the present study was to verify the favorable effects of SGB on the proliferation and function of splenic CD4 + T cells isolated from rats that underwent hemorrhagic shock and to investigate the mechanism related to the SGB interaction with autophagy and posthemorrhagic shock mesenteric lymph (PHSML). Male rats underwent SGB or sham SGB and conscious acute hemorrhage followed by resuscitation and multiple treatments. After 3 h of resuscitation, splenic CD4 + T cells were isolated to measure proliferation and cytokine production following stimulation with ConA in vitro. CD4 + T cells isolated from normal rats were treated with PHSML drained from SBG-treated rats, and proliferation, cytokine production, and autophagy biomarkers were detected. Hemorrhagic shock reduced CD4 + T cell proliferation and production of interleukin (IL)-2, IL-4, and tumor necrosis factor-α-induced protein 8-like 2 (TIPE2). SGB or administration of the autophagy inhibitor 3-methyladenine (3-MA) normalized these indicators. In contrast, administration of rapamycin (RAPA) autophagy agonist or intravenous injection of PHSML inhibited the beneficial effects of SGB on CD4 + T cells from hemorrhagic shocked rats. Furthermore, PHSML incubation decreased proliferation and cytokine production, increased LC3 II/I and Beclin-1 expression, and reduced p-PI3K and p-Akt expression in normal CD4 + T cells. These adverse effects of PHSML were also abolished by 3-MA administration, as well as incubation with PHSML obtained from SGB-treated rats. SGB improves splenic CD4 + T cell function following hemorrhagic shock, which is related to the inhibition of PHSML-mediated autophagy.

Therapeutic mechanisms of mesenchymal stem cells in acute respiratory distress syndrome reveal potentials for Covid-19 treatment.

The mortality rate of critically ill patients with acute respiratory distress syndrome (ARDS) is 30.9% to 46.1%. The emergence of the coronavirus disease 2019 (Covid-19) has become a global issue with raising dire concerns. Patients with severe Covid-19 may progress toward ARDS. Mesenchymal stem cells (MSCs) can be derived from bone marrow, umbilical cord, adipose tissue and so on. The easy accessibility and low immunogenicity enable MSCs for allogeneic administration, and thus they were widely used in animal and clinical studies. Accumulating evidence suggests that mesenchymal stem cell infusion can ameliorate ARDS. However, the underlying mechanisms of MSCs need to be discussed. Recent studies showed MSCs can modulate immune/inflammatory cells, attenuate endoplasmic reticulum stress, and inhibit pulmonary fibrosis. The paracrine cytokines and exosomes may account for these beneficial effects. In this review, we summarize the therapeutic mechanisms of MSCs in ARDS, analyzed the most recent animal experiments and Covid-19 clinical trial results, discussed the adverse effects and prospects in the recent studies, and highlight the potential roles of MSC therapy for Covid-19 patients with ARDS.

Estradiol-induced inhibition of endoplasmic reticulum stress normalizes splenic CD4 + T lymphocytes following hemorrhagic shock.

The aim is to investigate that 17ß-estradiol (E2)/estrogen receptors (ERs) activation normalizes splenic CD4 + T lymphocytes proliferation and cytokine production through inhibition of endoplasmic reticulum stress (ERS) following hemorrhage. The results showed that hemorrhagic shock (hemorrhage through femoral artery, 38-42 mmHg for 90 min followed by resuscitation of 30 min and subsequent observation period of 180 min) decreased the CD4+ T lymphocytes proliferation and cytokine production after isolation and incubation with Concanavalin A (5 µg/mL) for 48 h, induced the splenic injury with evidences of missed contours of the white pulp, irregular cellular structure, and typical inflammatory cell infiltration, upregulated the expressions of ERS biomarkers 78 kDa glucose-regulated protein (GRP78) and activating transcription factor 6 (ATF6). Either E2, ER-α agonist propyl pyrazole triol (PPT) or ERS inhibitor 4-Phenylbutyric acid administration normalized these parameters, while ER-ß agonist diarylpropionitrile administration had no effect. In contrast, administrations of either ERs antagonist ICI 182,780 or G15 abolished the salutary effects of E2. Likewise, ERS inducer tunicamycin induced an adverse effect similarly to that of hemorrhagic shock in sham rats, and aggravated shock-induced effects, also abolished the beneficial effects of E2 and PPT, respectively. Together, the data suggest that E2 produces salutary effects on CD4+ T lymphocytes function, and these effects are mediated by ER-α and GPR30, but not ER-ß, and associated with the attenuation of hemorrhagic shock-induced ERS.

Transcriptome Analysis Revealed Inflammation Is Involved in the Impairment of Human Umbilical Vein Endothelial Cells Induced by Post-hemorrhagic Shock Mesenteric Lymph.

Vascular endothelial injury caused by post-hemorrhagic shock mesenteric lymph (PHSML) return is an important manifestation during refractory hemorrhagic shock. Using human umbilical vein endothelial cells (HUVECs) and transcriptome analysis, this study sought to investigate the molecular mechanism underlying the adverse effect of PHSML on vascular endothelium. Post-hemorrhagic shock mesenteric lymph was collected from male rats after they underwent hemorrhagic shock and following resuscitation, while normal mesenteric lymph (NML) was harvested from sham rats. Human umbilical vein endothelial cells were incubated with the culture medium containing either 10% phosphate buffered saline (Control), NML, or PHSML for 3 h, and then were harvested for RNA sequencing. In comparison with NML treated cells, 37 genes were differentially expressed in PHSML-treated HUVECs, including 32 upregulated genes and five downregulated genes. These differentially expressed genes were mainly enriched in inflammatory pathways, including signaling pathways for activation of the NOD-like receptors, NF-κB, and TNF. Furthermore, we found that C-C motif chemokine ligand 2 (CCL2) was increased significantly after PHSML treatment, and Bindarit, a CCL2 production inhibitor, attenuated the damage of HUVECs induced by PHSML. The results provide molecular evidence on vascular endothelium damage caused by PHSML. C-C motif chemokine ligand 2 might represent a new target for reducing vascular injury after severe hemorrhagic shock.

Stellate Ganglion Blockade repairs Intestinal Mucosal Barrier through suppression of Endoplasmic Reticulum Stress following Hemorrhagic Shock.

Background: Hemorrhagic shock-induced ischemia and hypoxia elicit endoplasmic reticulum stress (ERS) that leads to cell apoptosis, tissue structural damage and organ dysfunction and failure. Stellate ganglion blockade (SGB) has been demonstrated to improve intestinal barrier dysfunction induced by hemorrhagic shock. The present study sought to investigate whether the beneficial effect of SGB on the intestinal mucosal barrier function is via suppression of ERS. Materials and methods: A conscious rat model of hemorrhagic shock (40 ±2 mmHg for 1 hour, followed by resuscitation) was established. The parameters reflecting intestinal morphology and intestinal mucosal barrier function including wet-dry ratio (W/D), intestinal permeability, D-lactic acid (D-LA) and intestinal fatty acid binding protein (I-FABP) in plasma, and expressions of ATF6α, PERK, and IRE1α in intestinal tissues were then observed. Furthermore, the effects of either SGB or ERS inhibitor, 4-phenylbutyric acid (4-PBA), on these parameters in rats with hemorrhagic shock were assessed. The effect of ERS agonist tunicamycin (TM) on the rats subjected with both SGB and hemorrhagic shock was also determined. Results: Either SGB or administration of ERS inhibitor, 4-PBA, alleviated hemorrhagic shock-induced adverse effects such as intestinal mucosal barrier dysfunction and excessive autophagy, which were characterized by damaged intestinal tissue, enhanced intestinal permeability and D-LA and I-FABP levels in plasma, and increased expressions of ATF6α, PERK, IRE1α in intestinal tissue. In contrast, administration of ERS agonist, TM, suppressed the beneficial effects of SGB on intestinal tissue and function during hemorrhagic shock. Conclusion: The SGB repairs intestinal mucosal barrier through suppression of ERS following hemorrhagic shock.

Engagement of Posthemorrhagic Shock Mesenteric Lymph on CD4+ T Lymphocytes In Vivo and In Vitro.

BACKGROUND: Immune dysfunction is associated with posthemorrhagic shock mesenteric lymph (PHSML) return. To determine the proliferation and cytokine production capacity of CD4+ T lymphocytes, the effect of PHSML drainage on spleen CD4+ T lymphocytes in a mouse model of hemorrhagic shock was assessed. METHODS: The normal spleen CD4+ T lymphocytes were in vitro incubated with either drained normal mesenteric lymph (NML), PHSML during hypotension (PHSML-H), or PHSML from 0 h to 3 h after resuscitation (PHSML-R) to verify direct proliferation effects of PHSML. RESULTS: Hemorrhagic shock led to reduction of proliferation and mRNA expression of interleukin 2 (IL-2) and IL-2 receptor in CD4+ T lymphocytes and to decrease in IL-2 and interferon γ (IFN-γ) levels in supernatants. In contrast, the interleukin-4 levels were increased. These effects were reversed by PHSML drainage. Moreover, NML incubation promoted CD4+ T lymphocyte proliferation, whereas both PHSML-H and PHSML-R treatment had a biphasic effects on CD4+ T lymphocyte proliferation, exhibiting an enhanced effect at early stages and an inhibitory effect at later stages. Compared with NML, PHSML-H increased IL-2 expression at 12 h, but decreased expression of both IL-2 and IFN-γ at 24 h. By contrast, PHSML-R induced significant increases in IL-2 and IFN-γ levels at 24 h. Interleukin-4 expression in CD4+ T lymphocytes was reduced at 12 h, but augmented at 24 h after incubation with either PHSML-H or PHSML-R. CONCLUSIONS: The results indicate that PHSML has a direct inhibitory effect on CD4+ T lymphocyte proliferation that induces an inflammatory response, which is associated with cellular immune dysfunction.

Blockade of Stellate Ganglion Remediates Hemorrhagic Shock-Induced Intestinal Barrier Dysfunction.

BACKGROUND: Acute hemorrhage-induced excessive excitation of sympathetic-adrenal-medullary system (SAS) leads to gut hypoperfusion and barrier dysfunction, which is a critical event during hemorrhagic shock-induced multiple organ injury. Stellate ganglion blockade (SGB) has been widely used for suppression of sympathetic-adrenal-medullary system in the clinical practice. However, whether SGB improves intestinal barrier function after hemorrhagic shock remains unclear. Here, we hypothesized that the implementation of SGB restores intestinal barrier function and reduces gut injury. MATERIALS AND METHODS: Male rats received the SGB pretreatment and underwent hemorrhagic shock followed by resuscitation. The 96-h survival rate, intestinal permeability and morphology, D-lactic acid concentration and diamine oxidase activity in plasma, and expressions of F-actin, Claudin-1, and E-cadherin in intestinal tissues were observed. RESULTS: Pretreatment with SGB significantly enhances the 96-h survival rate in rats subjected to hemorrhagic shock (from 8.3% to 66.7%). Hemorrhagic shock reduced the coverage scale of intestinal mucus and intestinal villus width and height, enhanced the intestinal permeability to fluorescein isothiocyanate-dextran 4 and D-lactic acid concentration in plasma, and decreased the expressions of F-actin, Claudin-1, and E-Cadherin in intestinal tissue. These hemorrhagic shock-induced adverse effects were abolished by SGB treatment. CONCLUSIONS: SGB treatment has a beneficial effect during hemorrhagic shock, which is associated with the improvement of intestine barrier function. SGB may be considered as a new therapeutic strategy for treatment of hemorrhagic shock.

Effect of Resveratrol on Blood Rheological Properties in LPS-Challenged Rats.

Objectives: Abnormal rheological properties induce adverse effects during sepsis. This study sought to investigate the hypothesis that resveratrol (Res) improves blood rheological properties in rats following a lipopolysaccharide (LPS) challenge, and provide a novel approach for treatment of sepsis. Methods: The rats were intraperitoneally or intramuscularly injected with vehicle, LPS (8 mg/kg), Res (30 mg/kg), or both to yield four groups: control, Res, LPS, and LPS + Res. After 6 h of LPS and/or Res injection, the mean arterial pressure (MAP), regional blood flow, erythrocyte and leukocyte parameters, and blood viscosity were observed. Results: LPS administration had no significant effects on the erythrocyte parameters and plasma viscosity. LPS administration reduced the MAP, whole blood viscosity at low and medium shear rates, the blood flow in the spleen and kidney, and the leukocyte content in whole blood when compared to control group, and increased the myeloperoxidase (MPO) activity in lung. Treatment with Res alone had no effects on most of parameters observed except increasing the whole blood relative viscosity. However, Res treatment after LPS resulted in further decrease in whole blood viscosity at high and medium shear rates. Furthermore, Res treatment conversely decreased the red blood cell distribution width-CV, blood flow of stomach, whole blood relative viscosity and MPO activity in lung, and increased the leukocyte content, but did not restore LPS-induced decrease in MAP and the blood flow in the spleen and kidney. Conclusion: The Res treatment partly reduce the whole blood viscosity and regional blood flow, and increase WBC content in peripheral blood following the LPS challenge, suggesting a favorable role in expanding the quasi-sympathetic effects of LPS in blood viscosity at early stages.

Resveratrol enhances vascular reactivity in mice following lipopolysaccharide challenge via the RhoA-ROCK-MLCP pathway.

The aim of the present study was to identify whether sepsis-induced vascular hyporeactivity is associated with microcirculation disturbance and multiple organ injuries. The current study assessed the impact of resveratrol (Res) treatment on lipopolysaccharide (LPS) challenge mediated vascular hyporeactivity. Effects of Res treatment (30 mg/kg; i.m.) at 1 h following LPS stimulation (5 mg/kg; i.v.) on the survival time, mean arterial pressure (MAP), and maximal difference of MAP (ΔMAP) to norepinephrine (NE; 4.2 µg/kg) in mice were observed. The reactivity to gradient NE of isolated mesenteric arterioles and the association with the RhoA-RhoA kinase (ROCK)-myosin light chain phosphatase (MLCP) pathway were investigated by myography, and the signaling molecule protein levels were assessed using ELISA. Res treatment prolonged the survival time of mice subjected to LPS challenge, but did not prevent the LPS-induced hypotension and increase in ΔMAP. Res treatment and RhoA agonist U-46619 incubation prevented LPS-induced vascular hyporeactivity ex vivo, which were suppressed by incubation with ROCK inhibitor Y-27632. LPS-induced vascular hyporeactivity was not affected by the MLCP inhibitor okadaic acid incubation, but was further downregulated by the co-incubation of OA plus Y-27632. The inhibiting effect of Y-27632 on Res treatment was eradicated by incubation with U-46619. Furthermore, RhoA inhibitor C3 transferase did not significantly inhibit the enhancing role of Res treatment, which was further increased by U-46619 plus C3 transferase co-incubation. In addition, Res treatment eradicated the LPS-induced decreases in p-RhoA and p-Mypt1 levels and increases in MLCP levels. The results of the present study indicate that post-treatment of Res significantly ameliorates LPS-induced vascular hyporeactivity, which is associated with the activation of the RhoA-ROCK-MLCP pathway.

[Role of mesenteric lymph drainage in the balance of ACE/ACE2 in murine myocardium following hemorrhagic shock].

OBJECTIVE: To observe the change of angiotensin converting enzyme (ACE) and ACE2 in the murine myocardium followed hemorrhagic shock and the role of post-hemorrhagic shock mesenteric lymph (PHSML) drainage. METHODS: Twenty-four male mice were ran-domly divided into control, sham, shock, and shock + drainage groups. A hemorrhagic shock model was established and then fluid resuscita-tion was performed to the mice in the shock and shock + drainage groups, and the PHMSL was drained in the shock + drainage group after fluid resuscitation. After 6 h of resuscitation in the shock and shock + drainage groups or corresponding time in the sham group, or after anesthesia in the control group, the myocardial tissues were harvested for the determination of the mRNA expressions of ACE, ACE2, angiotensin Ⅱ (Ang Ⅱ) type 1 receptor (AT1R), and Mas related G protein coupled receptor (Mas1R) using the method of qRT-PCR, and the levels of Ang Ⅱ and Ang (1-7) using the method of ELISA. RESULTS: In the myocardial tissue of shock group, the ACE and AT1R mRNA expressions and Ang Ⅱ level were significantly increased than those of the control and sham groups, the ACE2 and Mas1R mRNA expressions were significantly de-creased than that of the control and sham groups, the Ang (1-7) level was decreased compared with the control group, the ratios of ACE/ACE2, Ang Ⅱ/Ang (1-7), and AT1R/Mas1R in the shock group were significantly increased than the control and sham groups. Meanwhile, PHSML drainage obviously suppressed the effects of hemorrhagic shock on these indices. CONCLUSIONS: Hemorrhagic shock up-regulated the ACE-Ang Ⅱ-AT1R axis, down-regulated the ACE2-Ang (1-7)-Mas1R axis, and induced the unbalance of ACE and ACE2 in myocardial tis-sue. PHSML drainage decreased the ACE-Ang Ⅱ-AT1R axis and increased the ACE2-Ang (1-7)-Mas1R axis, resulted in the balance of ACE and ACE2.

Posthemorrhagic shock mesenteric lymph enhances monolayer permeability via F-actin and VE-cadherin.

BACKGROUND: Vascular hyperpermeability plays a critical role in the development of refractory hypotension after severe hemorrhagic shock. Posthemorrhagic shock mesenteric lymph (PHSML) return has been shown to be involved in regulation of vascular hyperpermeability. The present study was conducted to investigate the effect of PHSML on permeability of endothelial cells in vitro. MATERIALS AND METHODS: A hemorrhagic shock model (40 ± 2 mm Hg for 90 min, followed by fluid resuscitation) was used for collection of PHSML. Two separated PHSMLs were collected from period 0-3 h (early) and period 3-6 h (late) after resuscitation and diluted into concentration of 4% or 10%. The human umbilical vein endothelial cells (HUVECs) were then treated with these PHSMLs for 6 h. The monolayer cellular permeability to FITC-albumin was observed by using the costar transwell system. The multiple approaches including scanning electron microscope, fluorescent cytochemistry staining, and Western blotting were also used to assess the changes in cellular morphologic and the expressions of F-actin and VE-cadherin. RESULTS: The treatments with either early or late PHSML resulted in morphologic injuries, increased cellular permeability, and decreased expression of F-actin in HUVECs. In contrast, only early PHSML, but not late PHSML, reduced the VE-cadherin expression. CONCLUSIONS: These results indicate that the PHSML in vitro increases the cellular permeability of HUVECs through suppression of F-actin and VE-cadherin.

Nitric Oxide Regulates The Lymphatic Reactivity Following Hemorrhagic Shock Through Atp-Sensitive Potassium Channel.

Lymphatic reactivity has been shown to exhibit a biphasic change following hemorrhagic shock, and nitric oxide (NO) is involved in this process. However, the precise mechanism responsible for NO regulation of the lymphatic reactivity along with the progression of hemorrhagic shock is unclear. Therefore, the present study was to investigate how NO participates in regulating the shock-induced biphasic changes in lymphatic reactivity and its underlying mechanisms. First, the expressions or contents of inducible NO synthase, nitrite plus nitrate, and elements of cAMP-PKA-KATP and cGMP-PKG-KATP pathway in thoracic ducts tissue were assessed. The results revealed that levels of nitrite plus nitrate, cAMP, cyclic guanosine monophosphate (cGMP), p-PKA, and p-PKG were increased gradually along with the process of shock. Second, the roles of cAMP-PKA-KATP and cGMP-PKG-KATP in NO regulating lymphatic response to gradient substance P were evaluated with an isolated lymphatic perfusion system. The results showed that the NOS substrate (L-Arg), PKA donor (8-Br-cAMP) decreased the reactivity of shock 0.5 h-lymphatics, and that the PKA inhibitor (H-89) and KATP inhibitor (glibenclamide) restrained the effects of L-Arg while glibenclamide abolished the effects of 8-Br-cAMP. Meanwhile, NOS antagonist (L-NAME), protein kinase G (PKG) inhibitor (KT-5823), and soluble guanylate cyclase inhibitor (ODQ) increased the reactivity of shock 2 h-lymphatics, whereas KATP opener (pinacidil) inhibited these elevated effects induced by either L-NAME, ODQ, or KT-5823. Taken together, these results indicate that NO regulation of lymphatic reactivity during shock involves both cAMP-PKA-KATP and cGMP-PKG-KATP pathways. These findings have potential significance for the treatment of hemorrhagic shock through regulating lymphatic reactivity.

Inhibitory effect of post-hemorrhagic shock mesenteric lymph drainage on the HMGB1 and RAGE in mouse kidney.

BACKGROUND: Excessively inflammatory response is one of mechanisms that underlie the acute kidney injury (AKI) induced by severe hemorrhagic shock, which could be ameliorated by post-hemorrhagic shock mesenteric lymph (PHSML) blockage. Recent studies demonstrate that high mobility group box 1 (HMGB1) and the receptor for advanced glycation end products (RAGE) are critical mediators of local inflammations. The present study was sought to investigate whether the PHSML drainage inhibits the HMGB1 and RAGE in mouse kidney to ameliorate the renal inflammatory responses. METHODS: A mouse hemorrhagic shock model (40 ± 2 mmHg for 90 min, fluid resuscitation for 30 min) was employed, and the PHMSL drainage was performed at the end of the resuscitation. After 3 h of resuscitation, the expressions of mRNA and protein for the renal HMGB1 and RAGE and the levels of interleukin (IL)-1ß and IL-18 were assessed by the real-time reverse transcription-polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. RESULTS: Hemorrhagic shock elicited significant increases in the mRNA expressions of HMGB1 and RAGE and in the protein expressions of HMGB1, RAGE, IL-1ß and IL-18 in kidney. The PHSML drainage abolished these potentiating effects. CONCLUSION: The present study demonstrates that PHSML blockade reduces the increased HMGB1 and RAGE and pro-inflammatory factors following hemorrhagic shock, suggesting that the PHSML elicits the inflammatory responses via enhancing the HMGB1 and RAGE production in the kidney.